The invention is related to the area of gene therapy. In particular it is related to the field of expression monitoring.
In the context of gene therapy, it is important to monitor the expression of the therapeutic transgene. The effectiveness of any genetically based therapy will be critically dependent on the kinetics of gene expression, i.e., the time of onset of gene expression and its rate of increase; the maximum level of expression and its persistence and decay; and residual long-term expression. Where the gene produces a soluble protein such as a cytokine or growth factor, gene expression often can be monitored by measuring the levels of the secreted gene product in peripheral blood. However, many therapeutic transgenes code for cell-associated proteins whose expression cannot be conveniently monitored in this way. Current approaches to monitoring the expression of cell-associated transgenes are unsatisfactory and rely on the direct sampling and immunohistochemical analysis of genetically modified tissues at various time-points following the therapeutic procedure. There is a need in the art for methods and materials that enable the monitoring of transgene expression without the requirement to sample and directly test genetically modified cells or tissues.
The invention contemplates a method of monitoring the production of a therapeutic polypeptide in a patient, comprising the steps of (a) administering to a patient in need of a therapeutic polypeptide a nucleic acid construct encoding a therapeutic polypeptide and a marker polypeptide; and (b) detecting the marker polypeptide which has been released into extracellular body fluid of the patient as an indication of the amount of therapeutic polypeptide produced from the nucleic acid construct. In some embodiments, the step of detecting is used as a qualitative or semi-quantitative test merely to determine the presence or absence of the marker peptide. The presence of the marker peptide is an indication that the transgene has been successfully delivered to the target cell, tissue, or organ of the patient. In some embodiments, the nucleic acid construct also encodes a protease-cleavable linker that is situated between the therapeutic polypeptide and the marker peptide.
The invention also provides a method for monitoring the expression of a transgene. The method comprises the steps of (a) transfecting a cell using a nucleic acid construct, (b) obtaining a biological fluid sample from a patient containing the transfected cell, and (c) quantifying a marker peptide in the sample. The nucleic acid construct comprises a transgene and a sequence encoding a marker peptide that is non-immunogenic and non-functional. The marker peptide is released from the transfected cell into extracellular body fluid of the patient. In some embodiments, the nucleic acid construct also comprises a sequence encoding a protease-cleavable linker which is positioned between the transgene and the sequence encoding a marker peptide. The level of expression of the transgene is monitored by quantifying the marker peptide in the biological fluid sample.
The invention also provides a method of monitoring the expression of a transgene in a patient, comprising the steps of: (a) transfecting a host cell ex vivo with a nucleic acid construct comprising a transgene and a sequence encoding a marker polypeptide; (b) introducing the transfected host cell into a patient; and (c) quantifying the amount of marker polypeptide which has been released into extracellular body fluid of the patient, whereby the amount of the marker polypeptide is used to monitor the level of expression of said transgene. In some embodiments, the nucleic acid construct also encodes a protease-cleavable linker which is positioned between the transgene and the coding sequence for the marker polypeptide.
The invention also provides a nucleic acid construct. The nucleic acid construct comprises a transgene and a sequence encoding a marker polypeptide that is released from the cell where it is produced into extracellular fluid. The marker polypeptide is non-immunogenic and non-functional. In some embodiments, the construct also comprises a sequence encoding a protease-cleavable linker. The fusion polypeptide encoded by the construct does not form part of a naturally occurring precursor polypeptide from which the polypeptide encoded by the transgene is released by proteolytic cleavage. In some embodiments, the transgene encodes a fusogenic polypeptide. The sequence encoding a protease-cleavable linker, if present, is positioned between the transgene and the sequence encoding a marker polypeptide.
The invention also provides a host cell comprising the nucleic acid construct of the preceding paragraph.
The invention also provides a kit for practicing the invention. The kit comprises the nucleic acid construct described above and one or more reagents for monitoring the release of the marker polypeptide.
The invention also provides a kit comprising a host cell transfected with the nucleic acid construct described above and one or more reagents for monitoring the release of the marker polypeptide.
These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a nucleic acid construct for transfecting a cell with a transgene. The construct contains a sequence that encodes a marker polypeptide which serves as a detectable marker and a sequence that encodes a protease-cleavable linker peptide. When the transgene is expressed, the marker polypeptide and the linker are co-expressed in like amount. The linker is cleaved during normal post-translational processing by endogenous proteases within the cell to release a stoichiometric amount (i.e., a proportionate amount) of the marker polypeptide from the transgene product. The marker polypeptide is released from the cell where it is synthesized into extracellular fluid and is detectable in blood or other easily obtainable biological fluid samples and can be used to monitor the level and kinetics of expression of the transgene in the transfected cell or tissue. In a variation of this embodiment, the construct does not encode a protease-cleavable linker, but instead the marker polypeptide is regulated by a different promoter from that which regulates the transgene. In yet another variation of this embodiment, the construct does not encode a protease-cleavable linker, but instead the construct is transcribed to a polycistronic mRNA which comprises a ribosome entry site between the transgene and the sequence encoding the marker polypeptide.
Another embodiment of the invention provides a method for monitoring the expression of a transgene. The method employs the nucleic acid construct described in the preceding paragraph. A cell that has been transfected with this construct expresses the transgene as a fusion protein containing a marker polypeptide which is secreted from the cell and can be detected in blood or other easily obtainable-biological fluid samples. The marker polypeptide is non-immunogenic and non-functional. The marker polypeptide is released from the cell where it is made into the extracellular fluid. The marker peptide serves only as a marker whose level and kinetics of expression parallel those of the transgene product.
Still another embodiment of the invention provides another method for monitoring the expression of a transgene. A cell that has been transfected with the nucleic acid construct described above is introduced into a patient who has been transfected with the same transgene. In this embodiment, the bulk of the target cells are transfected with the transgene but not transfected with the marker polypeptide or linker of this invention; the cell which has been transfected with the construct of this invention, including the transgene, the marker polypeptide, and the linker, is used merely for expression monitoring of the transgene and is present only in sufficient amount to allow detection of the marker polypeptide. The marker polypeptide is released from the transfected cell into the extracellular fluid and serves as an indicator of transgene expression. In a variation of this embodiment, the cell is transfected with a construct that does not encode a protease-cleavable linker. Instead, the marker peptide is regulated by a different promoter from that which regulates the transgene. In another variation of this embodiment, the cell is transfected with a construct that is transcribed to a polycistronic mRNA which comprises an internal ribosome entry site between the transgene and the sequence encoding the marker peptide. Because of the position of the ribosome entry site, both the transgene product and the marker peptide are expressed separately without the need for protease cleavage.
Yet another embodiment of the invention provides a method of monitoring a therapeutic transgene. In this embodiment, the nucleic acid construct of this invention is used to transfect a cell as per either of the two previous embodiments. In this case, the transgene is a therapeutic gene which is introduced into the patient to remedy a functional deficiency, treat a pathological condition, or destroy certain cells of the patient by the activity of the transgene product. The marker polypeptide released from the transfected cell is detected and the information obtained is used to gage the progress of therapy with the transgene. In some versions of this embodiment, a transgene product which destroys cancer cells is monitored as a means of assessing the effectiveness of the therapy and deciding whether to repeat or adjust the therapy.
The invention thus provides the art with methods and materials for conveniently and effectively monitoring the level and kinetics of expression of transgenes in cells, tissues, animals or human patients without the need for disruptive and expensive sampling methods including surgery.